Materials and methods of using engineered ligands

Abstract

Described herein are compositions and kits that comprise an engineered TL1A ligand that displays high stability, minimal binding to decoy receptor DcR3 while retaining functional activity via binding to its cell surface receptor, DR3, and the ability to activate T cells in vitro and in vivo. Methods of making an engineered TL1A ligand and methods of treating a disease or disorder in a subject by administering an engineered TL1A ligand are also provided.

What is claimed is: 
     
       1. An engineered TNF-like factor 1A (TL1A) ligand, wherein the engineered TL1A ligand comprises
 a. at least one amino acid alteration of the amino acid sequence of SEQ ID NO:94 selected from the group consisting of K111, Q167, and E190 or; 
 b. at least one amino acid alteration of the amino acid sequence of SEQ ID NO:94 selected from the group consisting of K111A, K111S, K111E, L123G, L123S, L123E, L123K, Q167A, S187A, S187L, S187K, S187D, E190G, E190F, N207A, N207F, N207S, N207K and N207E, 
 and wherein the engineered TL1A ligand comprises a trimeric complex comprising: 
 a. three TL1A monomers, wherein the three TL1A monomers form a non-covalent TL1A trimer; or 
 b. three TL1A monomers, wherein the three TL1A monomers are covalently linked to form a single-chain TL1A (scTL1A) trimer. 
 
     
     
       2. The engineered TL1A ligand of  claim 1 , further comprising a protein stabilizing region, wherein optionally the protein stabilizing region comprises an Fc region, or a human serum albumin (HSA) region. 
     
     
       3. The engineered TL1A ligand of  claim 1 , comprising:
 a. the non-covalent TL1A trimer and one or more Fc regions; 
 b. the non-covalent TL1A trimer and one or more HSA regions; 
 c. the scTL1A trimer and one or more Fc regions; or 
 d. the scTL1A trimer and one or more HSA regions. 
 
     
     
       4. The engineered TL1A ligand of  claim 3 , comprising:
 a. two non-covalent TL1A trimers and three Fc regions; 
 b. two scTL1A trimers and one Fc region; 
 c. one scTL1A trimer and one Fc region; 
 d. one non-covalent TL1A trimer and three HSA regions; or 
 e. one scTL1A trimer and one HSA region. 
 
     
     
       5. The engineered TL1A ligand of  claim 1 , wherein the three TL1A monomers are covalently bound by a linker, wherein optionally the linker is a peptide linker, and wherein optionally the linker has an amino acid sequence of Gly-Ser or multiple repeats thereof. 
     
     
       6. The engineered TL1A ligand of  claim 1 , wherein the Fc region is a human IgGI, IgG2 or IgG4 Fc region, and wherein optionally non-covalent TL1A trimer or the scTL1A trimer is fused to the C-terminus of the Fc region. 
     
     
       7. The engineered TL1A ligand of  claim 1 , wherein:
 (i) the engineered TL1A ligand comprises the amino acid sequence of any one of SEQ ID NO:1-93; or 
 (ii) the engineered TL1A ligand comprises:
 a. an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identical over its entire length to the amino acid sequence of SEQ ID NO:79; 
 b. an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identical over its entire length to the amino acid sequence of SEQ ID NO:72; 
 c. an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identical over its entire length to the amino acid sequence of SEQ ID NO:8; 
 d. an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identical over its entire length to the amino acid sequence of SEQ ID NO:65; 
 e. an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identical over its entire length to the amino acid sequence of SEQ ID NO:52; 
 f. an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identical over its entire length to the amino acid sequence of SEQ ID NO: 14; 
 g. an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identical over its entire length to the amino acid sequence of SEQ ID NO:36; 
 h. an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identical over its entire length to the amino acid sequence of SEQ ID NO:90; 
 i. an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identical over its entire length to the amino acid sequence of SEQ ID NO:88; 
 j. an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identical over its entire length to the amino acid sequence of SEQ ID NO:91; or 
 k. an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identical over its entire length to the amino acid sequence of SEQ ID NO:89. 
 
 
     
     
       8. The engineered TL1A ligand of  claim 1 , (i) which comprises a bispecific antibody; (ii) which is fused to a heterologous polypeptide; or (iii) which is conjugated to an agent, wherein optionally the agent is a toxin. 
     
     
       9. The engineered TL1A ligand of  claim 1  comprising: a first means capable of binding DR3 with an affinity comparable to or higher than the affinity of wildtype TL1A and a second means capable of binding DcR3 with an affinity lower than the affinity of wildtype TL1A, wherein optionally:
 (i) the engineered TL1A ligand has a longer serum half-life than wildtype TL1A; 
 (ii) the engineered TL1A ligand has a high monodispersity and/or stability compared to wildtype TL1A; 
 (iii) the engineered TL1A ligand co-stimulates T cells in vitro; 
 (iv) the engineered TL1A ligand co-stimulates T cells in a subject; and/or 
 (v) the engineered TL1A ligand increases production of one or more cytokines in a subject, wherein optionally the one or more cytokines comprise IFNy and TNFa, wherein optionally the subject has an autoimmune disorder or cancer optionally selected from the group consisting of ulcerative colitis, lupus, inflammatory bowel disease (IBD), chronic obstructive pulmonary disease (COPD), arthritis, multiple sclerosis, diabetes, transplant rejection, central nervous system injury, Crohn's disease, psoriasis, leukemia or lymphoma, atherosclerosis, colon cancer, breast cancer, pancreatic cancer, leukemia, lung cancer such as non-small cell lung cancer, glioblastoma, melanoma, prostate cancer, gastric cancer, pituitary adenomas, ovarian cancer, renal cancer, bladder cancer, and a sarcoma, wherein optionally the sarcoma is a rhabdomyosarcoma, and wherein optionally the subject is a subject in need thereof. 
 
     
     
       10. A nucleic acid encoding the engineered TL1A ligand of  claim 1 . 
     
     
       11. A pharmaceutical composition, comprising the engineered TL1A ligand of  claim 1  or the nucleic acid encoding the engineered TL1A ligand, and a pharmaceutically acceptable excipient. 
     
     
       12. A method of making an engineered TL1A ligand comprising (i) a step for performing the function of introducing at least one amino acid alteration of the amino acid sequence of SEQ ID NO:94 selected from the group consisting of: K111A, L123K, M158Y, Q167A, S187L, E190F, and N207F; and (ii) a step for performing the function of producing a population of engineered TL1A ligand, wherein optionally the method further comprises the step of fusing the engineered TL1A ligand to a heterologous polypeptide, wherein optionally the heterologous polypeptide comprises a protein stabilizing region, and wherein optionally the protein stabilizing region comprises an Fc region, or a HSA region. 
     
     
       13. The method of  claim 12 , further comprising a step of generating a multimeric engineered TL1A ligand, wherein optionally:
 (i) the multimeric engineered TL1A ligand comprises:
 a. the non-covalent TL1A trimer and one or more Fc regions; 
 b. the non-covalent TL1A trimer and one or more HSA regions; 
 c. the scTL1A trimer and one or more Fc regions; or 
 d. the scTL1A trimer and one or more HSA regions, or 
 
 (ii) the multimeric engineered TL1A ligand comprises:
 a. two non-covalent TL1A trimers and three Fc regions; 
 b. two scTL1A trimers and one Fc region; 
 c. one scTL1A trimer and one Fc region; 
 d. one non-covalent TL1A trimer and three HSA regions; or 
 e. one scTL1A trimer and one HSA region. 
 
 
     
     
       14. The method of  claim 12 , wherein the engineered TL1A ligand comprises the amino acid sequence of any one of SEQ ID NO: 1-93, or wherein the engineered TL1A ligand comprises:
 a. an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identical over its entire length to the amino acid sequence of SEQ ID NO:79; 
 b. an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identical over its entire length to the amino acid sequence of SEQ ID NO:72; 
 c. an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identical over its entire length to the amino acid sequence of SEQ ID NO:8; 
 d. an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identical over its entire length to the amino acid sequence of SEQ ID NO:65; 
 e. an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identical over its entire length to the amino acid sequence of SEQ ID NO:52; 
 f. an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identical over its entire length to the amino acid sequence of SEQ ID NO: 14; 
 g. an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identical over its entire length to the amino acid sequence of SEQ ID NO:36; 
 h. an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identical over its entire length to the amino acid sequence of SEQ ID NO:90; 
 i. an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identical over its entire length to the amino acid sequence of SEQ ID NO:88; 
 j. an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identical over its entire length to the amino acid sequence of SEQ ID NO:91; or 
 k. an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identical over its entire length to the amino acid sequence of SEQ ID NO:89. 
 
     
     
       15. The method of  claim 12 , wherein the engineered TL1A ligand comprises three TL1A monomers each comprising amino acids 84 to 251 of SEQ ID NO:94 with K111A, L123K, Q167A, S187L, E190F and N207F mutations. 
     
     
       16. The engineered TL1A ligand of  claim 1 , wherein the engineered TL1A ligand comprises three TL1A monomers each comprising amino acids 84 to 251 of SEQ ID NO:94 with K111A, L123K, Q167A, S187L, E190F and N207F mutations. 
     
     
       17. The method of  claim 12  wherein the engineered TL1A ligand comprises amino acid alterations of K111A, L123K, Q167A, S187L, E190F and N207F of amino acid sequence SEQ ID NO:94. 
     
     
       18. The engineered TL1A ligand of  claim 1 , wherein the engineered TL1A ligand comprises amino acid alterations of K111A, L123K, Q167A, S187L, E190F and N207F of amino acid sequence SEQ ID NO:94.